Method for constructing bone microenvironment model through 3D printing and application

Abstract

The invention discloses a method for constructing a bone microenvironment model through 3D printing and application, and belongs to the field of three-dimensional tissue culture. The method comprises the following steps: printing bio-ink into a 3D printed stent based on a 3D printing technology; bone cells and collagen hydrogel are mixed, and a cell matrix material is constructed; and combining the cell matrix material with the 3D printing scaffold, incubating and curing, and then performing cell culture to form the bone microenvironment model. The in-vitro bone microenvironment model is constructed by a method of combining a 3D printing technology and a matrix material, and the model is applied to in-vitro construction of breast cancer and prostate cancer bone metastasis microenvironment models, so that the defects of in-vivo and in-vitro research methods of skeleton-related diseases are overcome; according to the method, the tissue structure and mechanical characteristics of a real bone microenvironment can be better simulated, and the constructed structure and function integrated in-vitro bone microenvironment model provides a more accurate platform for exploring bone tissue related diseases.

Description

Technical Field

[0001] The present invention relates to the field of three-dimensional tissue culture, and in particular to a method and application of constructing a bone microenvironment model through 3D printing. Background Art

[0002] Bone-related diseases are one of the major diseases that threaten human life and health. According to the World Health Organization (WHO), about 171 million people worldwide suffer from bone system diseases, of which 568 million suffer from low back pain, which is one of the main causes of disability. In China, the incidence of bone system diseases increased significantly between 1990 and 2019, with approximately 7%-10% of the population suffering from related diseases.

[0003] At present, the main research methods for bone-related diseases mainly rely on a combination of in vitro and in vivo experiments. In vitro experiments mostly focus on the cellular level, and common methods include the use of adherent cell culture, spheroid culture, and the construction of organoid models. In vitro experiments cannot meet the complex internal environment requirements of the body's complex multi-cell, multi-system, and multi-omics, and the types of cells that meet the requirements of in vitro culture are limited, and the bone microenvironment cannot be simulated, so it cannot be applied to a variety of bone-related diseases. In in vivo studies, experimental animals are a common tool for studying bone-related diseases. They have a normal bone microenvironment, but due to species differences, model stability, ethical issues and other factors, the accuracy of animal models in the application of bone-related diseases is limited. In order to deeply study the biological mechanisms of bone-related diseases, it is urgent to construct an in vitro bone microenvironment model that can simulate the tissue structure and mechanical characteristics of the real bone microenvironment and integrate structure and function.

[0004] 3D printing technology, also known as additive manufacturing technology, is a manufacturing technology that creates three-dimensional objects by stacking materials layer by layer. Unlike traditional methods of removing materials (such as cutting and milling), 3D printing is based on digital three-dimensional models and forms objects by continuously adding materials. At present, in the field of additive manufacturing of in vitro 3D printed bionic tissue models, relying on the high precision of 3D printing, 3D printing is combined with cells and other matrix materials to manufacture bionic extracellular microenvironment models. This method has been reported in tumor models and other disease models.

[0005] Although the currently reported 3D printed disease models can effectively preserve the activity and function of cells, the construction of a 3D printed bone microenvironment model in vitro is an unresolved problem due to the complex organic and inorganic environment of bone tissue.

[0006] The construction of a 3D printed bone microenvironment model can provide a more accurate platform for exploring bone tissue-related diseases, provide a new means for the clinical treatment of orthopedic diseases, and ultimately promote personalized treatment for patients and improve their treatment outcomes. Summary of the invention

[0007] The purpose of the present invention is to provide a method and application of constructing a bone microenvironment model by 3D printing to solve the problems existing in the above-mentioned prior art. The method of constructing an in vitro bone microenvironment model is achieved by combining 3D printing technology and matrix materials, and the model is applied to construct an in vitro breast cancer and prostate cancer bone metastasis microenvironment model to solve the shortcomings of in vivo and in vitro research methods for bone-related diseases.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method and application of constructing a bone microenvironment model by 3D printing, comprising the following steps:

[0010] Based on 3D printing technology, bio-ink is printed into 3D printed scaffolds;

[0011] Mixing bone cells with collagen hydrogel to construct cell matrix materials;

[0012] The cell matrix material is combined with the 3D printed scaffold, incubated and solidified, and then cell culture is performed to form a bone microenvironment model.

[0013] The present invention also provides a method for constructing a tumor bone metastasis microenvironment model by 3D printing, comprising the following steps:

[0014] Based on 3D printing technology, bio-ink containing cells and scaffold materials is printed into 3D printed scaffolds;

[0015] Mixing cells, tumor cells and collagen hydrogel to construct cell matrix materials;

[0016] The cell matrix material is combined with the 3D printed scaffold, incubated and solidified, and then cell culture is performed to form a tumor bone metastasis microenvironment model.

[0017] Preferably, the cells include at least one of osteoblast-related cells, osteoclast-related cells, immune cells, vascular endothelial cells, stem cells and mesenchymal cells; and / or the scaffold material includes at least one of bone cement, bioceramic materials (calcium phosphate ceramics, zirconium dioxide and aluminum dioxide), metal materials (calcium phosphate ceramics, zirconium dioxide and aluminum dioxide) and polymer materials (polylactic acid, polyetheretherketone and polycaprolactone), and can also be a composite material (ceramic-polymer composite material, metal-ceramic composite material); and / or the mass concentration of the scaffold material is 0.001%-20%, and the number of the cells is 1×10 2 -10×10 7 indivual.

[0018] Preferably, the osteoblast-related cells include at least one of bone mesenchymal stem cells, pre-osteoblastic cell lines and osteoblastic cell lines; and / or the osteoclast-related cells include monocytes-macrophages.

[0019] Preferably, the tumor cells include at least one of prostate cancer cells, breast cancer cells, lung cancer cells, thyroid cancer cells, urinary system tumor cells, gastrointestinal system tumor cells or reproductive system tumor cells; and / or the collagen hydrogel includes at least one of type I collagen hydrogel, hyaluronic acid hydrogel, sodium alginate hydrogel, gelatin hydrogel and decellularized matrix hydrogel.

[0020] Preferably, the combining method includes (1) covering the cell matrix material onto the 3D printed scaffold, or (2) printing the 3D printed scaffold on the cell matrix material; and / or the incubation and curing temperature is 35-40°C.

[0021] Preferably, the printing parameters of the 3D printed bracket are: size 0.1-50mm×0.1-50mm, line spacing 0.01-100mm, layer height 0.01-100mm, number of layers 2-50 layers, printing speed 0.1-100mm / s, and extrusion speed 0.01-50mm / s.

[0022] Preferably, 1-5 mL of collagen hydrogel is mixed with 1×10 2 -10×10 7 The cell matrix material is constructed by mixing individual cells.

[0023] Preferably, 1×10 2 -10×10 7 cells, 1×10 2 -10×10 7 The cell matrix material is constructed by mixing 100-10,000 tumor cells and 1-5 mL of type I collagen hydrogel; or by mixing 100-10,000 cell microspheres, 1-10,000 tumor cell microspheres and 1-5 mL of type I collagen hydrogel.

[0024] The present invention also provides an application of the tumor bone metastasis microenvironment model constructed by the method in any of the following items:

[0025] (1) Application in screening drugs for tumor bone metastasis;

[0026] (2) Application in screening radiotherapy plans.

[0027] The present invention discloses the following technical effects:

[0028] The present invention constructs an in vitro bone microenvironment model by combining 3D printing technology and matrix materials, and applies the model to construct in vitro breast cancer and prostate cancer bone metastasis microenvironment models. The feasibility of in vitro construction of the bone microenvironment model is evaluated through in vitro experiments and animal experiments. The in vitro tumor bone metastasis microenvironment model has good clinical and scientific research application effects, which makes up for the shortcomings of in vivo and in vitro research methods of bone-related diseases, and is of great significance for the treatment and clinical intervention of bone-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 Immunofluorescence staining diagram of the 3D printed bone microenvironment model provided by the present invention; A is a diagram of the immunofluorescence staining results of COL and RUNX-2 of cells carried by the 3D printed bone microenvironment model, and B is a diagram of the immunofluorescence staining results of F-actin of cells carried by the 3D printed bone microenvironment model;

[0031] Figure 2 A confocal fluorescence image of the prostate cancer bone metastasis microenvironment model provided by the present invention;

[0032] Figure 3 This is a fluorescence image of the CTX treatment of the 231-HMCAF cell microspheres provided by the present invention in the model. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The tumors involved in the present invention include, but are not limited to, one or a combination of prostate cancer, breast cancer, lung cancer cells, thyroid cancer cells, urinary system tumor cells, gastrointestinal system tumor cells, and reproductive system tumor cells. In the embodiments of the present invention, prostate cancer and breast cancer are used as examples for illustration.

[0039] The "osteoblast-related cells" involved in the present invention are MSCs, which can be one or a combination of cells with osteogenic differentiation ability that are considered feasible by those skilled in the art, such as pre-osteoblast cell line and osteoblast cell line.

[0040] The "osteoclast-related cells" involved in the present invention are primary monocyte-macrophages, which can be one or a combination of cells with osteoclast differentiation ability that are considered feasible by those skilled in the art, such as RAW264.7.

[0041] The osteoblast and osteoclast-related cells involved in the present invention are derived from mice. They may be selected from rats or humans, but are not limited thereto. They may be one or a combination of osteoblast and osteoclast species that are considered feasible by those skilled in the art. The present invention is described using mouse-derived osteoblast and osteoclast-related cells as an example.

[0042] The "carrying cells" involved in the present invention are preferably osteoblast-related cells and osteoclast-related cells, and can also be one or a combination of immune cells, vascular endothelial cells, related stem cells, and related mesenchymal cells. However, it is not limited thereto. Those skilled in the art believe that all cells present in the tumor bone metastasis microenvironment can be carried on the 3D printed scaffold.

[0043] The "bio-ink" involved in the present invention is preferably calcium phosphate bone cement and starch-modified calcium phosphate bone cement, and optionally, also includes one or a combination of derivative materials with calcium phosphate bone cement (solid phase: hydroxyapatite and two or more calcium salts of α-tricalcium phosphate, β-tricalcium phosphate, and dihydrate calcium hydrogen phosphate, liquid phase: distilled water, phosphoric acid, and saline) and acrylate bone cement (solid phase: polymethyl methacrylate, liquid phase: monomer methyl acrylate) as substrates.

[0044] The "type I collagen" involved in the present invention is selected from the rat type I collagen of ibidi in the United States.

[0045] The "hyaluronic acid" involved in the present invention is preferably human, with a molecular weight of 500-300000. The hyaluronic acid can be selectively modified with thiol groups, or unmodified. The present invention is described using unmodified hyaluronic acid as an example.

[0046] The "sodium alginate" involved in the present invention includes low viscosity, or one or a mixture of low viscosity and medium-high viscosity. In the embodiments of the present invention, low-viscosity sodium alginate is used as an example for explanation.

[0047] The "matrix material" involved in the present invention is preferably type I collagen hydrogel, which can be hyaluronic acid hydrogel, sodium alginate hydrogel, gelatin hydrogel, but the gel is not limited to one or a combination of the above-mentioned gel types, including gels that are considered feasible by those skilled in the art. In the embodiment of the present invention, sodium alginate hydrogel is selected for illustration.

[0048] The "matrix material" involved in the present invention may also be added with one or a combination of the following components: antimicrobial peptides, cytokines, inflammatory factors, growth factors, serum-related proteins, and immune cells, vascular endothelial cells, related stem cells, related mesenchymal cells, osteoclast-related cells, etc. that relevant technicians in the field believe to exist in the bone microenvironment.

[0049] The matrix material of the present invention preferably carries cells, but may not carry cells.

[0050] The osteogenic-related cells carried by the matrix material of the present invention are preferably bone mesenchymal stem cells (MSC), and may also be one or a combination of cells with osteogenic differentiation ability that are considered feasible by those skilled in the art, such as pre-osteoblastic cell lines and osteoblastic cell lines.

[0051] The prostate cancer cells carried by the matrix material of the present invention are preferably PC3, and can also be one or a combination of prostate cancer cells considered feasible by technicians in this field, such as LNCaP, DU145, 22Rv1, VCaP and primary prostate cancer cells.

[0052] The matrix material of the present invention is preferably loaded with cell microspheres, but may not be loaded with cell microspheres.

[0053] The cell microspheres carried by the matrix material of the present invention are preferably constructed with 100-10000 231-RFP and 100-10000 HMCAF-GFP. The breast cancer cells can be one or a combination of breast cancer cells considered feasible by those skilled in the art, such as MCF-7, T47D, BT-474, MDA-MB-468, SK-BR-3, ZR-75-1 and primary breast cancer cells. It may or may not contain breast cancer-related fibroblasts.

[0054] The combination method of the matrix material and the 3D printing bracket involved in the present invention is preferably direct covering, and 3D printing can also be performed in the matrix material.

[0055] The following is an example of the construction and cultivation of a bone microenvironment model carrying osteoblast-related cells and osteoclast-related cells, but is not limited to this. Those skilled in the art believe that all cells present in the bone microenvironment can be carried on the model as part of the bone microenvironment model of the present invention.

[0056] Example 1 A method for constructing a bone microenvironment model using 3D printing

[0057] In this embodiment, osteoblast- and osteoclast-related cells are added to the 3D printed scaffold of the bone metastasis microenvironment model to simulate the bone matrix microenvironment, and osteoblast-related cells are added to the matrix material to simulate the bone marrow matrix microenvironment. The above can simulate the bone microenvironment more realistically.

[0058] The bone microenvironment model was constructed by 3D printing, and then osteogenic induction of MSC and osteoclast induction of primary monocyte-macrophages were performed. The MSCs and primary monocyte-macrophages used were purchased from the ATCC cell bank. Immunofluorescence staining was used to evaluate the osteogenic and osteoclastogenic induction effects of the cell-loaded bone microenvironment model after induction. The specific steps are as follows:

[0059] 1) 3D printing process: Under sterile conditions, the bio-ink is mixed with 1% w / v calcium phosphate bone cement and sterile water in proportion until uniform, and the bio-ink is loaded into a syringe with a 24G needle. During printing, the temperature of the bone cement is controlled at 15°C, and then a filamentous mesh structure is formed by extrusion through the needle. The printing parameters are as follows: the design size of the 3D mesh structure is 10mm×10mm, line spacing: 5mm, layer height: 5mm, number of layers: 15 layers, printing speed: 2.5mm / s, extrusion speed: 1mm / s. After printing, the mesh structure is placed in 37°C for incubation and curing. Then 6×10 3 Osteogenesis-related cells (osteoblastic cell line) and 2×10 5 Osteoclast-related cells (RAW264.7) were seeded onto the grid structure and cultured in complete medium.

[0060] 2) Construction of bone microenvironment model: First, construct the cell-laden matrix material. Under sterile conditions, 5×10 5 Osteogenesis-related cells (MSCs) were mixed with 3 mL of type I collagen hydrogel (1.2 mg / mL type I collagen, 0.5 mg / mL hyaluronic acid, 5 mg / mL sodium alginate). The preparation process was carried out on ice. Then, the above matrix material was covered on the 3D printed grid structure, and the composite structure was placed in an incubator for incubation and solidification. The bone microenvironment model was cultured using complete culture medium.

[0061] 3) Cell culture: After 1 hour, add cell culture medium to the bone microenvironment model and continue to culture in the incubator. After 1 day of culture, the printed bone microenvironment model can be subjected to osteogenic and osteoclast induction interventions, using mouse osteoblast induction medium (Zhongqiao Xinzhou, PCM-I-002) and mouse osteoclast induction medium (Punosai, PD-028-100) for induction culture. After 7 days of culture, the cells can be immunofluorescently stained for type I collagen (COL), Runt-related transcription factor 2 (RUNX-2), and fibrous actin (F-actin).

[0062] The experimental results of the construction of the 3D printed bone microenvironment model are as follows Figure 1 The immunofluorescence staining results show that the model has the functions of osteogenic and osteoclastogenic induction.

[0063] Example 2 A method for constructing a bone microenvironment model using 3D printing

[0064] In this embodiment, osteoblast- and osteoclast-related cells are added to the 3D printed scaffold of the bone metastasis microenvironment model to simulate the bone matrix microenvironment, and osteoblast-related cells are added to the matrix material to simulate the bone marrow matrix microenvironment. The above can simulate the bone microenvironment more realistically.

[0065] The bone microenvironment model was constructed by 3D printing, and then osteogenic induction of MSC and osteoclast induction of primary monocyte-macrophages were performed. The MSCs and primary monocyte-macrophages used were purchased from the ATCC cell bank. Immunofluorescence staining was used to evaluate the osteogenic and osteoclastogenic induction effects of the cell-loaded bone microenvironment model after induction. The specific steps are as follows:

[0066] 1) 3D printing process: Under sterile conditions, the bio-ink is mixed with 0.1% w / v calcium phosphate bone cement and sterile water in proportion until uniform, and the bio-ink is loaded into a syringe with a 23G needle. During printing, the temperature of the bone cement is controlled at 15°C, and then a filamentous mesh structure is formed by extrusion through the needle. The printing parameters are as follows: the design size of the 3D mesh structure is 3mm×3mm, line spacing: 0.1mm, layer height: 0.1mm, number of layers: 2 layers, printing speed: 1mm / s, extrusion speed: 0.1mm / s. After printing, the mesh structure is placed in 37°C for incubation and curing. Then 5×10 2 Osteogenesis-related cells (osteoblast line) and 3×10 4 - 3×105 osteoclast-related cells (RAW264.7) were seeded onto the grid structure and the cells were cultured using complete medium.

[0067] 2) Construction of bone microenvironment model: First, construct the cell-laden matrix material. Under sterile conditions, 3×10 4 The osteoblast cell line was mixed with 1 mL of type I collagen hydrogel (1 mg / mL type I collagen, 0.1 mg / mL hyaluronic acid, 1 mg / mL sodium alginate). The preparation process was carried out on ice. Then, the above matrix material was covered on the 3D printed grid structure, and the composite structure was placed in an incubator for incubation and solidification. The bone microenvironment model was cultured using complete culture medium.

[0068] 3) Cell culture: After 1 hour, add cell culture medium to the bone microenvironment model and continue to culture in the incubator. After 1 day of culture, the printed bone microenvironment model can be subjected to osteogenic and osteoclast induction interventions, using mouse osteoblast induction medium (Zhongqiao Xinzhou, PCM-I-002) and mouse osteoclast induction medium (Punosai, PD-028-100) for induction culture. After 7 days of culture, the cells can be immunofluorescently stained for type I collagen (COL), Runt-related transcription factor 2 (RUNX-2), and fibrous actin (F-actin).

[0069] The results of immunofluorescence staining were the same as those in Example 1, and the constructed model also had the functions of osteogenic induction and osteoclastogenic induction.

[0070] Example 3 A method for constructing a bone microenvironment model using 3D printing

[0071] In this embodiment, osteoblast- and osteoclast-related cells are added to the 3D printed scaffold of the bone metastasis microenvironment model to simulate the bone matrix microenvironment, and osteoblast-related cells are added to the matrix material to simulate the bone marrow matrix microenvironment. The above can simulate the bone microenvironment more realistically.

[0072] The bone microenvironment model was constructed by 3D printing, and then osteogenic induction of MSC and osteoclast induction of primary monocyte-macrophages were performed. The MSCs and primary monocyte-macrophages used were purchased from the ATCC cell bank. Immunofluorescence staining was used to evaluate the osteogenic and osteoclastogenic induction effects of the cell-loaded bone microenvironment model after induction. The specific steps are as follows:

[0073] 1) 3D printing process: Under sterile conditions, the bio-ink is mixed with 5% w / v calcium phosphate bone cement and sterile water in proportion until uniform, and the bio-ink is loaded into a syringe with a 25G needle. During printing, the temperature of the bone cement is controlled at 15°C, and then a filamentous mesh structure is formed by extrusion through the needle. The printing parameters are as follows: the design size of the 3D mesh structure is 30mm×30mm, line spacing: 10mm, layer height: 10mm, number of layers: 20 layers, printing speed: 5mm / s, extrusion speed: 2mm / s. After printing, the mesh structure is placed in 37°C for incubation and curing. Then 8×10 3 Osteogenesis-related cells (osteoblasts) and 3×10 5 Osteoclast-related cells (RAW264.7) were seeded onto the grid structure and cultured in complete medium.

[0074] 2) Construction of bone microenvironment model: First, construct the cell-laden matrix material. Under sterile conditions, 8×10 5 The osteoblast cell line was mixed with 1-5 mL of type I collagen hydrogel (1.5 mg / mL type I collagen, 1 mg / mL hyaluronic acid, 10 mg / mL sodium alginate). The preparation process was carried out on ice. Then, the above matrix material was covered on the 3D printed grid structure, and the composite structure was placed in an incubator for incubation and solidification. The bone microenvironment model was cultured using complete culture medium.

[0075] 3) Cell culture: After 1 hour, add cell culture medium to the bone microenvironment model and continue to culture in the incubator. After 1 day of culture, the printed bone microenvironment model can be subjected to osteogenic and osteoclast induction interventions, using mouse osteoblast induction medium (Zhongqiao Xinzhou, PCM-I-002) and mouse osteoclast induction medium (Punosai, PD-028-100) for induction culture. After 7 days of culture, the cells can be immunofluorescently stained for type I collagen (COL), Runt-related transcription factor 2 (RUNX-2), and fibrous actin (F-actin).

[0076] The results of immunofluorescence staining were the same as those in Example 1, and the constructed model also had the functions of osteogenic induction and osteoclastogenic induction.

[0077] Application example: A method and application of 3D printing to construct a tumor bone metastasis microenvironment model

[0078] Taking prostate cancer and breast cancer as examples, a tumor bone metastasis microenvironment model is constructed, which can be used for the differentiation of the above-mentioned prostate cancer or breast cancer stem cells and the manufacture of an in vitro bone metastasis tumor organoid model. The above-mentioned prostate cancer or breast cancer cells are derived from humans.

[0079] 1. Construction of a prostate cancer bone metastasis microenvironment model and its application in radiotherapy and chemotherapy treatment

[0080] This application example adds osteoblast-related cells to the 3D printed scaffold of the bone metastasis microenvironment model, and adds osteoblast-related cells and prostate cancer cells to the matrix material. This can more realistically simulate the situation of prostate cancer in the bone metastasis microenvironment and the inhibitory effect of osteoblast-related cells on prostate cancer cells in vivo. Therefore, it can be used for the screening of anticancer drugs for prostate cancer.

[0081] A prostate cancer bone metastasis microenvironment model was constructed by 3D printing, and then the model was used for chemotherapy drug screening experiments. The screening effect was then evaluated by immunofluorescence staining, confocal fluorescence photography, and other means. The MSCs, preosteoblast cell lines (MC3T3E1S14, MC3T3), and prostate cancer cells (PC3) used were purchased from the ATCC cell bank. The control group: cultured with complete medium; the drug-added group: cultured with complete medium containing enzalutamide (XTANDI). The above model was photographed using a confocal fluorescence microscope. The specific steps are as follows:

[0082] 1) 3D printing process: Under sterile conditions, the bio-ink is mixed in proportion with 2.5% w / v starch-modified calcium phosphate bone cement and saline until uniform, and the bio-ink is loaded into a syringe with a 24G needle. During printing, the temperature of the bone cement is controlled at 15°C, and then a filamentous mesh structure is formed by extrusion through the needle. The printing parameters are as follows: the design size of the 3D mesh structure is 15mm×15mm, line spacing: 20mm, layer height: 20mm, number of layers: 20 layers, printing speed: 2.5mm / s, extrusion speed: 1mm / s. After printing, the mesh structure is placed in 37°C for incubation and curing. Then 6×10 3 Osteogenesis-related cells (pre-osteoblast cell line) were seeded onto the grid structure and cultured using complete culture medium.

[0083] Preparation of the above-mentioned starch-modified calcium phosphate bone cement: First, tricalcium phosphate and dicalcium hydrogen phosphate dihydrate powders are uniformly mixed in a ratio of 9:1, and the obtained mixed powder is freeze-dried to obtain calcium phosphate bone cement powder. Then, barium sulfate, starch and calcium phosphate cement powder are mixed in a mass ratio of 2:2:6 to obtain starch-modified calcium phosphate bone cement powder. Finally, the starch-modified calcium phosphate bone cement powder is mixed with physiological saline or phosphate buffer to obtain 2.5% w / v starch-modified calcium phosphate bone cement.

[0084] 2) Construction of bone microenvironment model: First, construct the cell-laden matrix material. Under sterile conditions, 5×10 5 Osteoblast-related cells (MSCs), 5×10 6 Prostate cancer cells (PC3-Luc) expressing luciferase reporter gene (Luc) and 3mL type I collagen hydrogel (1.2mg / mL type I collagen, 0.5mg / mL hyaluronic acid, 5mg / mL sodium alginate, 20% Matrigel matrix gel) were mixed. The preparation process was carried out on ice. Then, the above matrix material was covered on the 3D printed grid structure, and the composite structure was placed in an incubator for incubation and solidification. The bone microenvironment model was cultured using complete culture medium.

[0085] 3) Cell culture: After 1 hour, add cell culture medium to the bone microenvironment model and continue to culture in the incubator. After 1 day of culture, mouse osteoclast induction medium (Punocet, PD-028-100) can be used for induction culture. After 7 days of culture, the XTANDI drug screening experiment can be performed on the 3D printed prostate cancer bone metastasis microenvironment model, and the screening method is the same as the traditional method. Confocal fluorescence photography experiments are performed on the prostate cancer bone metastasis microenvironment model before and after treatment to evaluate the screening effect.

[0086] 4) Anticancer drug screening experiment: XTANDI was used as an anticancer drug to test its sensitivity to PC3 cell microspheres. First, a complete medium containing XTANDI was prepared at a concentration of 1 μL / mL. The prostate cancer bone metastasis microenvironment model was then cultured with complete medium without or with XTANDI, and the medium was replaced every two days. Confocal fluorescence photography was performed on the PC3 cell microspheres in the model on the first, seventh, and fourteenth days of culture.

[0087] The experimental results of drug screening in the 3D printed prostate cancer bone metastasis microenvironment model are as follows Figure 2 shown. Figure 2 This is a confocal fluorescence image of the prostate cancer bone metastasis microenvironment model. The results show that XTANDI can effectively inhibit the growth of PC3 cell microspheres in the prostate cancer bone metastasis microenvironment model, indicating that this model has application prospects in clinical and basic research.

[0088] 2. Construction of breast cancer bone metastasis microenvironment model and its application in chemoradiotherapy model

[0089] This application example adds osteoblast- and osteoclast-related cells into the 3D printed scaffold of the bone metastasis microenvironment model and adds breast cancer cell microspheres into the matrix material, which can more realistically simulate the situation of breast cancer in the bone metastasis microenvironment. Therefore, it can be used to screen anticancer drugs for breast cancer and verify the effects of different radiotherapy dose regimens, and test the effects of radiotherapy on cancer cells and normal breast tissue.

[0090] A breast cancer bone metastasis microenvironment model was constructed by 3D printing, and then the model was subjected to drug intervention experiments and radiotherapy intervention experiments in vitro. The breast cancer cells (MDA-MB-231, 231) and breast cancer-associated fibroblasts (HMCAF) used were purchased from the ATCC cell bank. Anticancer drugs were screened in the breast cancer bone metastasis microenvironment model, and a control group was set up: cultured with complete culture medium; a drug-added group: cultured with complete culture medium containing cyclophosphamide (CTX). The above model was photographed using a confocal fluorescence microscope, and the specific operation steps are as follows:

[0091] 1) Preparation of matrix material: First, construct a cell-carrying matrix material. Under sterile conditions, mix 1-1000 231-RFP cells (5000 cells / microsphere) expressing red fluorescent protein (RFP) and HMCAF-GFP cells (5000 cells / microsphere) expressing green fluorescent protein (GFP) with 3 mL of type I collagen hydrogel (1.2 mg / mL type I collagen, 0.5 mg / mL hyaluronic acid, 5 mg / mL sodium alginate, 2% Matrigel matrix gel). The preparation process is carried out on ice. Then, the above matrix material is placed in a 4°C environment for 3D printing.

[0092] 2) Specific process of 3D printing: Under sterile conditions, the bio-ink is made by mixing 2% w / v calcium phosphate bone cement and saline in proportion until uniform. The bio-ink also carries 5×10 7 osteoblast-related cells (MSCs) and 5×10 7 Osteoclast-related cells (primary monocytes-macrophages) were added, and the bio-ink was loaded into a syringe with a 24G needle. The temperature of the bone cement was controlled at 15°C during printing, and then a filamentous mesh structure was formed by extrusion through the needle. The printing parameters are as follows: the design size of the 3D mesh structure is 6mm×6mm, line spacing: 10mm, layer height: 10mm, number of layers: 8 layers, printing speed: 1mm / s, and extrusion speed: 1mm / s. After printing, the mesh structure printed in the matrix material was incubated at 37°C for solidification, and the cells were cultured using complete culture medium.

[0093] 3) Cell culture: After 1 hour, add cell culture medium to the bone microenvironment model and continue to culture it in the incubator. After 1 day of culture, the printed bone microenvironment model can be intervened by osteogenic and osteoclast induction, and induced culture can be performed using mouse osteoblast induction medium (Zhongqiao Xinzhou, PCM-I-002) and mouse osteoclast induction medium (Punose, PD-028-100). After 1 day of culture, the 3D printed breast cancer bone metastasis microenvironment model can be used for anticancer drug screening or radiotherapy screening, and the screening and intervention methods are the same as the traditional methods. Radiotherapy intervention experiments on breast cancer bone metastasis microenvironment models use radiotherapy of different doses and frequencies. Confocal fluorescence photography of the breast cancer bone metastasis microenvironment model was performed before and after treatment.

[0094] 4) Anticancer drug screening experiment: CTX was used as an anticancer drug to test its sensitivity to 231 breast cancer cell microspheres. First, a complete medium containing CTX was prepared at a concentration of 5 μg / mL. The prostate cancer bone metastasis microenvironment model was then cultured with complete medium without or with CTX, and the medium was replaced every two days. Confocal fluorescence photography of the 231 breast cancer cell microspheres in the model was performed on the first, seventh, and fourteenth days of culture.

[0095] 5) Radiotherapy screening experiment: The model was tested using 30Gy / 10 and 10Gy / 10 radiotherapy regimens. 30Gy / 10 radiotherapy regimen: The radiation dose for each radiotherapy was 3Gy, with a total of 10 irradiations, which was completed within an average of 2 weeks. 10Gy / 10 radiotherapy regimen: The radiation dose for each radiotherapy was 1Gy, with a total of 10 irradiations, which was completed within an average of 2 weeks. Confocal fluorescence photography of the 231 breast cancer cell microspheres in the model was performed on the first, seventh, and fourteenth days of radiotherapy.

[0096] The experimental results of drug screening in the 3D printed breast cancer bone metastasis microenvironment model are as follows Figure 3 shown. Figure 3 This is a fluorescence image of 231-HMCAF cell microspheres treated with CTX in the model, and the results show that CTX can effectively inhibit the growth of 231 cell microspheres in the breast cancer bone metastasis microenvironment model, indicating that the model has application prospects in clinical and basic research.

[0097] Similarly, the experimental results of radiotherapy screening for the 3D printed breast cancer bone metastasis microenvironment model showed that a radiotherapy regimen of 30Gy / 10 times could effectively inhibit the growth of 231 cell microspheres, indicating that the model has application prospects in clinical and basic research.

[0098] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method and application of constructing a bone microenvironment model by 3D printing, characterized in that: The following steps are involved: Based on 3D printing technology, bio-ink is printed into 3D printed scaffolds; Mixing bone cells with collagen hydrogel to construct cell matrix materials; The cell matrix material is combined with the 3D printed scaffold, incubated and solidified, and then cell culture is performed to form a bone microenvironment model.

2. A method for constructing a tumor bone metastasis microenvironment model by 3D printing, characterized in that: The following steps are involved: Based on 3D printing technology, bio-ink containing cells and scaffold materials is printed into 3D printed scaffolds; Mixing cells, tumor cells and collagen hydrogel to construct cell matrix materials; The cell matrix material is combined with the 3D printed scaffold, incubated and solidified, and then cell culture is performed to form a tumor bone metastasis microenvironment model.

3. The method according to claim 1 or 2, characterized in that The cells include at least one of osteoblast-related cells, osteoclast-related cells, immune cells, vascular endothelial cells, stem cells and mesenchymal cells; and / or the scaffold material includes at least one of bone cement, bioceramic material, metal material and high molecular polymer material; and / or the mass concentration of the scaffold material is 0.001%-20%, and the number of the cells is 1×10 2 -10×10 7 indivual.

4. The method according to claim 3, characterized in that The osteoblast-related cells include at least one of bone mesenchymal stem cells, pre-osteoblastic cell lines and osteoblastic cell lines; and / or the osteoclast-related cells include monocyte-macrophages.

5. The method according to claim 1 or 2, characterized in that: The tumor cells include at least one of prostate cancer cells, breast cancer cells, lung cancer cells, thyroid cancer cells, urinary system tumor cells, gastrointestinal system tumor cells or reproductive system tumor cells; and / or the collagen hydrogel includes at least one of type I collagen hydrogel, hyaluronic acid hydrogel, sodium alginate hydrogel, gelatin hydrogel and decellularized matrix hydrogel.

6. The method according to claim 1 or 2, characterized in that: The combining method includes (1) covering the cell matrix material onto the 3D printed scaffold, or (2) printing the 3D printed scaffold on the cell matrix material; and / or the incubation and curing temperature is 35-40°C.

7. The method according to claim 1 or 2, characterized in that: The printing parameters of the 3D printed bracket are: size 0.1-50mm×0.1-50mm, line spacing 0.01-100mm, layer height 0.01-100mm, number of layers 2-50, printing speed 0.1-100mm / s, and extrusion speed 0.01-50mm / s.

8. The method according to claim 1, characterized in that 1-5 mL collagen hydrogel with 1×10 2 -10×10 7 The cell matrix material is constructed by mixing individual cells.

9. The method according to claim 2, characterized in that 1×10 2 -10×10 7 cells, 1×10 2 -10×10 7 The cell matrix material is constructed by mixing 100-10,000 tumor cells and 1-5 mL of type I collagen hydrogel; or by mixing 100-10,000 cell microspheres, 1-10,000 tumor cell microspheres and 1-5 mL of type I collagen hydrogel.

10. Use of the tumor bone metastasis microenvironment model constructed by the method according to claim 2 in any of the following: (1) Application in screening drugs for tumor bone metastasis; (2) Application in screening radiotherapy plans.

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